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PDBsum entry 1utm
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Trypsin specificity as elucidated by lie calculations, X-Ray structures, And association constant measurements.
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Authors
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H.K.Leiros,
B.O.Brandsdal,
O.A.Andersen,
V.Os,
I.Leiros,
R.Helland,
J.Otlewski,
N.P.Willassen,
A.O.Smalås.
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Ref.
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Protein Sci, 2004,
13,
1056-1070.
[DOI no: ]
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PubMed id
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Abstract
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The variation in inhibitor specificity for five different amine inhibitors bound
to CST, BT, and the cold-adapted AST has been studied by use of association
constant measurements, structural analysis of high-resolution crystal
structures, and the LIE method. Experimental data show that AST binds the 1BZA
and 2BEA inhibitors 0.8 and 0.5 kcal/mole more strongly than BT. However,
structural interactions and orientations of the inhibitors within the S1 site
have been found to be virtually identical in the three enzymes studied. For
example, the four water molecules in the inhibitor-free structures of AST and BT
are channeled into similar positions in the S1 site, and the nitrogen atom(s) of
the inhibitors are found in two cationic binding sites denoted Position1 and
Position2. The hydrophobic binding contributions for all five inhibitors,
estimated by the LIE calculations, are also in the same order (-2.1 +/- 0.2
kcal/mole) for all three enzymes. Our hypothesis is therefore that the observed
variation in inhibitor binding arises from different electrostatic interactions
originating from residues outside the S1 site. This is well illustrated by AST,
in which Asp 150 and Glu 221B, despite some distance from the S1 binding site,
lower the electrostatic potential of the S1 site and thus enhance substrate
binding. Because the trends in the experimentally determined binding energies
were reproduced by the LIE calculations after adding the contribution from
long-range interactions, we find this method very suitable for rational studies
of protein-substrate interactions.
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Figure 1.
Figure 1. Structural formulas of the synthetic trypsin
inhibitors included in the study. The numbers 1-5 refer roughly
to the strength of the inhibitors.
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Figure 2.
Figure 2. Structural arrangements, 2F[o]-F[c] (cyan)
electron density maps and Fourier difference maps (Fo-F[c]) at
+4  (green) and
-4 (red) of the
active site of AST (A-D) and BT (E-H). The inhibitors are (A)
benzamidine AST-1BZA, (B) benzylamine AST-2BEA, (C)
phenylethylamine AST-3PEA, (D) phenylpropylamine AST-4PPA, (E)
aniline BT-ANL, (F) benzylamine BT-2BEA, (G) phenylethylamine
BT-3PEA, and (H) phenylbutylamine BT-5PBA. Ser 190 is not
covered by electron density and only selected hydrogen bonds are
included to simplify the figure created by BobScript (Esnouf
1997). The sigma levels of the 2F[o]-F[c] maps are 1.1-1.5 (AST)
and 1.5-1.7 (BT).
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The above figures are
reprinted
by permission from the Protein Society:
Protein Sci
(2004,
13,
1056-1070)
copyright 2004.
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